Antibiotic—Lysobacter enzymogenes proteases combination as a novel virulence attenuating therapy

Minimizing antibiotic resistance is a key motivation strategy in designing and developing new and combination therapy. In this study, a combination of the antibiotics (cefixime, levofloxacin and gentamicin) with Lysobacter enzymogenes (L. enzymogenes) bioactive proteases present in the cell- free supernatant (CFS) have been investigated against the Gram-positive methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA) and the Gram-negative Escherichia coli (E. coli O157:H7). Results indicated that L. enzymogenes CFS had maximum proteolytic activity after 11 days of incubation and higher growth inhibitory properties against MSSA and MRSA compared to E. coli (O157:H7). The combination of L. enzymogenes CFS with cefixime, gentamicin and levofloxacin at sub-MIC levels, has potentiated their bacterial inhibition capacity. Interestingly, combining cefixime with L. enzymogenes CFS restored its antibacterial activity against MRSA. The MTT assay revealed that L. enzymogenes CFS has no significant reduction in human normal skin fibroblast (CCD-1064SK) cell viability. In conclusion, L. enzymogenes bioactive proteases are natural potentiators for antimicrobials with different bacterial targets including cefixime, gentamicin and levofloxacin representing the beginning of a modern and efficient era in the battle against multidrug-resistant pathogens.


Introduction
Infectious diseases are one of the leading causes of illness and death among individuals worldwide [1]. A wide variety of infections can be caused by either primary or opportunistic pathogens [2] which can acquire and transfer resistance genetically by either mutation or gene transfer thus, microbes control became a challenge [3]. The World Health Organization (WHO) has considered the emergence of antibiotic-resistant bacterial strains as one of the Most clinically relevant antibiotics are derived from natural products (actinomycetes or fungi), their semisynthetic isolates or synthetic [5]. The mining of these antibiotics commonly resulted in the discovery of similar compounds, suggesting an urgent need for a shift in attention to utilize previously uncharacterized microbes as a source of novel antibiotics. A relevant number of plant-beneficial bacteria are effective biocontrol agents against plant pathogenic microorganisms and are referred to as 'green' biopesticides [6]. These microorganisms played an important role in increasing crop production by protecting plants from infectious pathogens, hence lowering the use of carcinogenic pesticides in agriculture [6,7]. Within the soil microbiome, the Lysobacter genus acquired high attention due to its role in controlling pathogen-induced plant diseases [8]. For example, Lysobacter enzymogenes (L. enzymogenes) C3 and OH11, were reported to be effective in controlling Bipolaris leaf-spot on tall fescue and anthracnose on pear fruit, caused by Bipolaris sorokiniana and Colletotrichum fructicola fungal pathogens, respectively [9,10]. Thus, Lysobacter species have been designated as facultative predators able to lyse several microorganisms including nematodes, bacteria and fungi [11,12] through epibiotic predation and cell-cell contact. Worth mentioning that the molecular mechanism involved in Lysobacter genus killing behavior, is still not fully explored. However, Xi Shen research group revealed that L. enzymogenes OH11 uses Type IV Secretion System (T4SS) as the main contact-dependent weapon against other soil-borne bacteria [13,14]. Other studies indicated that L. enzymogenes bacteriolytic activity is attributed to its extracellular alpha (a serine protease) and beta (metalloprotease) lytic enzymes [15]. These proteases together with other enzymes such as chitinases, glucanases, lipases and phospholipases can degrade the cell wall of some plant pathogens including Gram-positive and Gram-negative bacteria [15][16][17]. Thereafter, Lysobacter has emerged as a new source of bioactive natural products [18,19] and attention has been pointed to their ability to lyse both prokaryotic and eukaryotic microbes with the production of peptides that damage microorganisms' cell walls or membranes at a very low concentration [20].
This study aims to investigate the potential implications of L. enzymogenes cell-free supernatant (CFS) bioactive products alone and in combination with cefixime, levofloxacin and gentamicin antibiotics against clinically important pathogens including staphylococcus aureus (S. aureus), one of the most notorious species causing mild and serious infections such as necrotizing pneumonia, septicemia and bone infections [41], Escherichia coli (E. coli) O157:H7, a well-described food-borne pathogen which produces virulence factors [42] and is responsible for bloody diarrhea and hemolytic-uremic syndrome (HUS) [43].
L. enzymogenes bacterial pellets were rehydrated aseptically using 5 mL of 10% strength tryptone soy broth (TSB), incubated at 28˚C and shaken at 200 rpm for 3 days in a shaker incubator (MS, Taiwan) [44]. Then, L. enzymogenes culture (200 μL) was seeded over 10% strength of tryptone soy agar (TSA) plate and incubated at 28˚C for 3 days. L. enzymogenes pure broth culture (PBC) was prepared by transferring a full loop from the stock plate to a 50 mL sterile falcon containing 10% TSB, followed by incubation at 28˚C for 3 days. E. coli O157:H7 was cultured in nutrient broth (NB) (Biolab); MSSA and MRSA in brain heart infusion (BHI) broth (Biolab).

Preparation of L. enzymogenes cell-free supernatant (CFS) bioactive products and proteolytic activity assessment
Two different pools of L. enzymogenes PBC (10 7 CFU/mL) were prepared and were assigned as pool A (PA, 4% (v/v)) and pool B (PB, 8% (v/v)). PA was prepared by mixing 0.5 mL of the PBC in a 15 mL falcon tube containing 12 mL 10% TSB and incubating the mixtures at 28˚C for different time intervals (1, 2, 3, 4, 7, 10, 11, 14 and 18 days) with continuous shaking at 200 rpm. PB was prepared using 1 mL of the PBC following the steps used for PA (S1 Fig). The plate count method was applied to enumerate the incubated PA and PB culture series viable cells. Later, L. enzymogenes cells of PA and PB cultures series were collected by centrifugation (15000 rpm at 4˚C for 15 min), filtration using a sterile syringe filter (0.22 μm) to obtain the cell-free supernatant (CFS) and stored at -20˚C in small aliquots for later use. The proteolytic activity of the obtained CFS series (Fig 1) was measured using Sigma's non-specific protease activity assay [45]. In this assay, L. enzymogenes proteases digest casein substrate to liberate free tyrosine which reacts with Folin's reagent (Folin and Ciocalteu's) to generate a blue color solution. The absorbance of this solution was measured at 660 nm using a microtiter plate reader (Epoch-Biotec, California, USA) and compared with the absorbance of different standard tyrosine concentrations (S2 Fig). All experiments were performed in triplicate.
L. enzymogenes CFS proteolytic activity was correlated with L. enzymogenes viable cell count and determined in terms of units/mL, which is corresponding to the micromoles (μmol) of tyrosine released from casein per minute, by applying the following equation [45].

Determination of antibiotics' minimum inhibitory concentrations (MIC)
The susceptibility of MSSA, MRSA and E. coli O157:H7 to the three antibiotics; cefixime, levofloxacin and gentamicin was performed using National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method [46]. Minimum inhibitory concentration (MIC) was determined using 96 flat-bottom microtiter plates (TPP, Switzerland). Each test well was filled with 90 μL BHI for S. aureus and NB for E. coli O157:H7. An aliquot (100 μL) of the antibiotic stock solution was added to the test well and mixed. A series of twelve 2-fold serial dilutions of the antibiotics were examined. The concentration ranges used to determine MICs were: cefixime 256-0.125 μg/mL, levofloxacin 64-0.031μg/mL, gentamicin 128-0.062 μg/mL against S. aureus and cefixime 32-0.0156 μg/mL, levofloxacin 32-0.0156 μg/mL, gentamicin 64-0.031 μg/mL against E. coli O157:H7. All dilutions of the tested antibiotics were inoculated with 10 μL of 10 6 CFU/mL of the specified bacterial strain and then, incubated at 37˚C for 24 h. Positive control (broth and bacterial suspension) and negative control (broth only) wells were included in every experiment to prove adequate microbial growth and media sterility during the incubation period.
In the test wells, microbial growth was assessed visually from culture turbidity and compared to the negative and positive controls. MICs were determined as the lowest concentration of the antibiotic that inhibits the growth of the microorganism. The test was carried out in triplicate (in the same 96-well plate) and repeated twice for each bacterium.

Growth inhibition activity of L. enzymogenes CFS, antibiotics and their combination
A microplate growth inhibition assay [47] was applied to measure the growth inhibitory effect of L. enzymogenes CFS, antibiotics (cefixime, levofloxacin and gentamicin) and their combination against MSSA, MRSA and E. coli O157:H7. This assay allows the observation of discernible inhibition during growth using turbidity parameter which is measured through the detection of light scatter in absorbance at 600 nm using a microplate reader.
For the evaluation of antibiotics' antibacterial activity, the MIC, 0.5 MIC, 0.25 MIC and 0.125 MIC concentrations for each antibiotic (cefixime, levofloxacin and gentamicin) were prepared. The test was carried out by placing 180 μL BHI for S. aureus and NB for E. coli O157:H7 and 10 μL of the prepared antibiotic dilution in each well, then 10 μL aliquot of the pathogen (10 6 CFU/ml) was added.
As a full growth control run, a 10 μL aliquot of the pathogenic cell suspension was inoculated at 10 6 CFU/mL into 190 μL of the corresponding sterile broth. Also, a test blank was run with each experiment where the 10 μL of the pathogen was replaced by the proper sterile broth.
In the above-mentioned experiments, the microtiter plate was incubated at 37˚C for 24 h and the optical density (OD) was measured at 600 nm using a microplate reader (Epoch-Biotec, California, USA). Results were calculated as the average mean of three readings.
In this study, antibacterial activity was expressed as percentage inhibition of bacterial growth following 24 h incubation at 37˚C and calculated using the following equation: Where: • OD T is the average optical density of three replicates at 600 nm for the tested target (L. enzymogenes CFS, antibiotics or the combination) • OD TB is the average optical density of three replicates at 600 nm for the tested target blank (10 μL of the pathogen is replaced by equivolume of the appropriate sterile broth) • OD FG is the average optical density of three replicates at 600 nm for the study pathogen full growth.
• OD FGB is the average optical density of three replicates at 600 nm for the study pathogen blank (No pathogen, only 10 μL of broth was used).

Cytotoxic activity
MTT assay was applied to evaluate L. enzymogenes CFS cytotoxicity. The cell line used in this assay was human normal skin fibroblast (CCD-1064SK) purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). This colorimetric assay measures the cellular metabolic activity based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the yellow 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide dye (MTT) to form the insoluble formazan purple crystals [48][49][50]. Cells were seeded in a 96-well culture plates in a final volume of 100 μL media per well, then plates were incubated in a humidified atmosphere (37˚C, 5% CO 2 ) for 24 h to allow cells to adhere. When cells reached confluency, they were treated with the L. enzymogenes CFS to obtain final concentrations of 1.25, 2.5, 5, 10, 20, 25, 40, and 50% (v/v). Cells were incubated in the humidified atmosphere (37˚C, 5% CO 2 ) for 72 h before performing the MTT assay to determine the cells' viability. The assay was conducted in triplicate, and the control cells were treated only with 10% TSB. The optical density for the treated and control wells was measured at 570 nm using a microtiter plate reader (BioTek, Winooski, VT, USA). Percentage viability was calculated using the formula below

Statistical analysis
Data were expressed as a mean ± standard deviation (SD). The statistical significance between different test conditions was determined using the independent sample t-test. The difference among groups was significant when p < 0.05.

L. enzymogenes cell-free supernatant (CFS) proteolytic activity assessment
L. enzymogenes CFS proteolytic activity was measured using Sigma's universal protease activity assay. L. enzymogenes CFS proteolytic activity was evaluated under two variables; bacterial density (PA, 4% (v/v) and PB, 8% (v/v)) and incubation period (1, 2, 3, 4, 7, 10, 11, 14 and18 days) as presented in Fig 1. Irrespective of the bacterial density (PA and PB), comparable proteolytic activity was observed during the first ten days of incubation ( Fig 1A). However, on day eleven, a change in proteolytic activity was detected. The maximal proteolytic activity for the PA and PB stocks were 0.129 unit/mL and 0.093 unit/mL (Fig 1A), respectively. Accordingly, higher inoculum size did not yield higher proteases production.
To gain insight into the characters of bioactive products secreted by L. enzymogenes, viable cell count of PA 4% (v/v) and PB 8% (v/v) stocks at different incubation periods was evaluated (Fig 1B). Results revealed that L. enzymogenes multiplication process was active during the first two incubation days, thereafter the growth rate decelerates due to depletion of the essential nutrients. Following incubation for eleven days, bacterial death for PA and PB cultures was dominant ( Fig 1B) and so it may be proposed that L. enzymogenes intracellular proteases were released signifying the maximum proteolytic activity observed (Fig 1A).

Bacterial inhibition capacity of L. enzymogenes CFS against MSSA and MRSA
L. enzymogenes CFS growth inhibitory effect was first screened against S. aureus. A reduction in MSSA and MRSA colonies density and size was visually noticed when L. enzymogenes CFS (100 and 200 μL) was flooded over a plate cultured with 10 8 CFU/mL of S. aureus as shown in Fig 2A and 2B.
The MICs of the standard antibiotics (cefixime, levofloxacin and gentamicin) on the selected pathogens are summarized in Table 1.
The antibacterial activity of the standard antibiotics (cefixime, gentamicin and levofloxacin) at 0.5 MIC (Fig 4A), 0.25 MIC (Fig 4B) and 0.125 MIC (Fig 4C) alone and in combination with L. enzymogenes CFS against MSSA, MRSA and E coli O157:H7 are presented in Fig 4. The combination of L. enzymogenes CFS with cefixime, gentamicin, levofloxacin at sub-MICs showed an additive effect against MSSA and MRSA (Fig 4). L. enzymogenes CFS-antibiotics 0.5 MICs mixtures presented fifty percent or higher growth inhibition activity against MSSA. Apparently, L. enzymogenes CFS triggered cefixime's antibacterial activity against MSSA when tested at its 0.5 MIC (p = 0.001, Fig 4A), 0.25 MIC (p = 0.004, Fig 4B) and 0.125 MIC (p = 0.045, Fig 4C). Yet, no significant additive effect for L. enzymogenes CFS when combined with cefixime against the Gram-negative E. coli O157:H7 at the sub-MIC values. On the contrary, gentamicin and levofloxacin combination at sub-MIC strength with L. enzymogenes CFS increased their antibacterial activity against all tested pathogens (Fig 5).

Discussion
Interest in antibiotic adjuvants therapy has increasingly attracted attention within contemporary studies due to the emergence of multidrug-resistant organisms [51][52][53]. Perhaps one of the leading causes of this resistance is the low microbial cell membrane permeability to antibiotics. Hence, microbial proteases able to perturb other pathogens membrane structure arises as an efficient tool to increase antibiotic bioavailability. Indeed, recent studies reported the   combination of outer-membrane acting peptides (natural or synthetic) with antibiotics inhibiting cell-wall synthesis as a new pathway for finding effective therapy [54,55]. L. enzymogenes, an environment-friendly soil-borne pathogen produces several proteolytic enzymes. Some are secreted into the culture medium while others are localized in the cellenvelope [15,18]. In the current study, maximum proteolytic activity for L. enzymogenes was obtained following PBC incubation for 11 days, during which the intracellular peptidases were released into the media following cell destruction (Fig 1). L. enzymogenes PBC PA (4% (v/v) superior proteolytic activity (1.3 folds) compared to PB (8% (v/v), could be attributed to the higher bacterial surface area to volume ratio, hence improving dissolved oxygen and nutrients' consumption ( Fig 1A).
L. enzymogenes CFS growth inhibitory potential was influenced by the strain and the concentration of the pathogen under investigation (Fig 3). L. enzymogenes CFS displayed higher inhibitory activity against S. aureus (MSSA and MRSA) compared to E. coli O157:H7 at the infectious tested bacterial concentration (Fig 3). This might be attributed to the core difference between Gram-positive and Gram-negative bacteria cell wall structure and composition [56]; S. aureus lack the outer lipopolysaccharide membrane enabling L. enzymogenes proteinases to perturb the multilayered peptidoglycan membrane, whilst in E. coli, L. enzymogenes proteinases have to cross the lipopolysaccharide layer in order to invade the monolayer peptidoglycan. As reported, L. enzymogenes α-Lytic protease specifically cleaves peptide bonds near small and hydrophilic amino acids as alanine, serine, threonine and valine, while β-lytic proteases specifically targets glycine, and also, the D-Ala-X bonds in bacterial cell wall peptidoglycan moieties [57]. Consequently, the higher bacteriolytic activity of L. enzymogenes CFS observed (Fig 3) toward S. aureus compared to E. coli O157:H7 could be attributed to its lower extent to disrupt the peptide-bridge cross-linking in E. coli O157:H7 peptidoglycan layer.
The decline in L. enzymogenes CFS antibacterial activity against higher bacterial concentration (Fig 3) could be related to the reduction in proteinases' concentration in the culture supernatant due to the binding of these proteinases with viable, lysed bacterial structures and chemical components. Thus, L. enzymogenes CFS percentage growth inhibitory activity is proportional to the amount of culture supernatant proteases available to each bacterium at the time of exposure.
Antimicrobials with different bacterial targets including cefixime (cell wall synthesis inhibitor) [58], levofloxacin (DNA gyrase blocker) [59] and gentamicin (protein synthesis inhibitor) [60] at sub-MIC levels in combination with L. enzymogenes CFS, is promising (Fig 5). Gentamicin and levofloxacin antibacterial activity at half, quarter and one eighth the MIC against the tested pathogens has been potentiated when combined with L. enzymogenes CFS (Fig 5). This cooperative effect might be related to the CFS proteolytic activity toward peptidoglycan moieties, hence facilitating the accessibility of gentamicin and levofloxacin to their intracellular targets.
The reported resistance of MRSA to cefixime [61] was interestingly overcome when combined with L. enzymogenes CFS. Herein, cefixime-L. enzymogenes CFS combination may be recommended as a new strategy to combat infectious diseases caused by β-Lactam resistant MRSA.
Microbial proteases, though essentially indispensable to the maintenance and survival of their host, can be potentially damaging when present in other hosts. Antimicrobial peptides are often limited by their cytotoxicity [62]. The in vitro cytotoxicity is quantitatively evaluated by MTT assay. This bioassay is based on the intracellular reduction of methyltetrazolium salt by the viable cells [63,64]. According to the International Organization for Standardization (ISO) 10993-5 [65], tested materials that reveal a reduction in cell viability by more than 30% are regarded as cytotoxic. The present research indicated high cell viability above 90% for all L. enzymogenes CFS tested concentrations. This finding supports the use of L. enzymogenes CFS with antibiotics as an adjuvant to treat bacterial infections. This combination presents a great potential for becoming a future strategy to achieve therapeutic goals. Yet, further research is required to move this therapy forward.

Conclusion
In summary, L. enzymogenes CFS and antibiotics combinations showed positive antibacterial activity against the Gram-positive S. aureus (MRSA and MSSA) and the Gram-negative pathogen E. coli O157:H7 as a new trend to combat bacterial resistance. L. enzymogenes CFS is a good potentiator for gentamicin and levofloxacin antibacterial activity, thus lowering the doses administered and hence reducing their side effects. In addition, cefixime's antibacterial spectrum against MRSA was recovered when combined with L. enzymogenes CFS. MTT assay revealed that L. enzymogenes CFS exhibited no significant reduction in cell viability against human normal skin fibroblast (CCD-1064SK). Given the promising results obtained so far, the appeal of using combinatory therapy will have great potential. It could represent the beginning of a modern and efficient era in the battle against multidrug resistant pathogens.